Morphological characterisation of portal myofibroblasts and hepatic stellate cells in the normal dog liver

Routine haematoxylin and eosin (H&E) sections in all dogs revealed a normal liver. With large individual differences, presumptive vitamin A-storing HSC were regularly seen with a single large vacuole (vitamin A-storing lipid droplet) and a dislocated nucleus. HSC without a vitamin A-storing vacuole ("empty HSC") could not be identified on H&E sections. In immunostaining, negative controls were negative.

There was strong variation between slides. In the portal area, vimentin showed positive staining cells in smooth muscle cells of portal vasculature, most spindle-shaped stromal cells and neural cells (Fig. 1a). Cells in Glisson's capsule as well as all stromal cells around the sublobular hepatic vein reacted positively to vimentin antibody. However, HSC were generally negative, although some individual positive cells were present (Fig. 1b).

There was also marked variation between slides. In general, HSC were weakly positive in the perinuclear cytoplasm, but vitamin A-storing HSC were predominantly negative (Fig. 2a). In the portal areas moderate to strong staining was present in the smooth muscle cells of the arterial tunica muscularis and in the perivenular smooth muscle cells of the portal vein (Fig. 2b). In addition, few positive spindle-shaped stromal cells were seen throughout the portal stroma and in the periductal location. In the sublobular hepatic veins smooth muscle cells were positive, in the surrounding stroma some MF appeared weakly positive. Incidentally, some positive cells were seen in Glisson's capsule.

Consistently in all slides, this marker showed a slightly irregular (1–3 μm wide) moderate staining in the perisinusoidal spaces throughout the hepatic parenchyma (Figs. 3a,3b), and with higher magnification positive staining cells were observed containing small lipid vacuoles consistent with HSC (Fig. 3c). Vitamin A-storing HSC mostly showed positive cytoplasmic staining. Around the terminal and sublobular hepatic veins positive staining of pericytes and smooth muscle cells was observed, endothelial cells were consistently negative. Portal areas showed strong positivity around the bile ducts and in the arterial tunica media, and moderate positivity in the wall of the portal veins, while endothelial cells remained negative (Fig. 3d). Some portal MF, particularly in larger portal triads, showed weak to moderate α-SMA positivity (Fig. 3d). Glisson's capsule showed few positive cells.

Staining for this marker generally rendered similar results as α-SMA, consistently in all slides. In the portal areas, the terminal and sublobular hepatic veins, and in Glisson's capsule identical staining was observed. In the hepatic parenchyma moderate positive staining was seen in the HSC (Figs. 4a,4b). In comparison with α-SMA, HHF35 accentuated the perinuclear cytoplasm. As with α-SMA, regularly positive staining cells were seen with 1 to 3 small cytoplasmic lipid vacuoles (Fig. 4a). However, cells with a single large vitamin A-storing vacuole were mostly negative (Fig. 4b).

In formalin fixed normal canine liver tissue GFAP staining revealed few positive nerves located in larger portal areas (internal positive control) but no other positive staining was observed in any other location in these sections. Despite strong staining for synaptophysin in the adrenal medulla (external positive control), no staining was observed in any of the formalin fixed normal liver sections. In the frozen chronic hepatitis case (with expected activated hepatic MF and HSC), only nerves in larger portal areas reacted positively to GFAP and NCAM, while synaptophysin did not provoke any signal at all.

Transmission electron microscopy (TEM) for α-SMA and HHF35 revealed a strong granular cytoplasmic staining restricted to subendothelial cells with long cytoplasmic extensions located in Disse's space (Figs. 5, 6). These cells often contained several smaller or one larger empty vacuole, interpreted as fat vacuoles (Fig. 5). Very slight, inevitable background staining was present in sinusoids and cells as small irregular spots, to be distinguished from the positive gold granules by a smaller and more irregular size. Both location and morphology of the positively staining cells identified them as HSC.

Normal liver tissue was obtained from ten dogs for immunohistochemistry: either patients with liver unrelated pathology (n = 8) or normal control animals euthanized for liver-unrelated research projects (n = 2). Laboratory exams regarding liver function were not performed. One frozen sample of a dog with chronic hepatitis was additionally used. Patients were submitted for their individual diagnostic purposes to the Department of Clinical Sciences of Companion Animals, or to the Department of Pathology, Faculty of Veterinary Medicine, Utrecht University. No tissue was taken purposely for the reported study. Projects were approved by the responsible ethical committees for the use of experimental animals and for use of client-owned animals according to Dutch legislation. After euthanasia as part of the research projects, we were allowed to take liver tissue of the two control animals. Included were six females and four males. Mean age was 13 months (± 15 months).

Liver specimens were taken within 1 hour post mortem (n = 9), or in a surgical biopsy procedure (n = 1). The normal liver samples were fixed in 10% neutral buffered formalin and routinely embedded in paraffin, while the chronic hepatitis sample was snap-frozen in liquid nitrogen cooled isopentane and stored at -70°C. Sections (3 μm) were stained with haematoxylin and eosin for routine histology. Immunohistochemistry was performed for α-SMA, HHF35, desmin, vimentin, GFAP and synaptophysin on all normal liver sections, the frozen sections (chronic hepatitis) were subjected to GFAP, NCAM and synaptophysin immunohistochemical staining. Antibody characteristics, manufacturer, source and dilution are given in Table 1. For this purpose, sections (3 μm) were mounted on poly-L lysine coated slides and stored for a maximum of 48 hours at room temperature until use. After that slides were deparaffinized. Endogenous peroxidase activity was blocked by 1% H₂O₂ in methanol for 30 min at RT. As the protocols for demonstration of desmin and synaptophysin required an antigen retrieval step [13, 21] sections were treated by heating in 10 mM citrate pH 6.0 in a microwave oven for 10 min, cooled down for 10 min at room temperature (RT). After washing with PBS buffer containing 0.1% Tween-20, background staining was blocked by incubating the sections with normal horse serum (1:10 diluted) for 15 min at RT for α-SMA, HHF35, synaptophysin and vimentin. Desmin and GFAP sections were blocked with normal goat serum (1:10 diluted) for 15 min at RT. Sections were incubated 60 min at RT with the primary antibody to α-SMA, desmin, GFAP or HHF35, and 30 min at RT for vimentin, while sections for synaptophysin were incubated overnight at 4°C. After washing in PBS-Tween, slides to be marked with mouse antibodies were incubated in horse-anti-mouse biotin (Vector Laboratories, Burlingame, CA, USA) (1:125 diluted) for 30 min at RT. GFAP and desmin sections were incubated in goat anti rabbit biotin (1:250 diluted) for 30 min at RT. After washing in PBS-Tween, sections were incubated in avidin-biotin peroxidase complex (Vector Laboratories). The colour was developed in 3-3'-diaminobenzidine, sections were counterstained with 10% Mayer's haematoxylin. Negative controls consisted of omission of the primary antibody, and replacement by non-immune serum. Formalin fixed paraffin embedded canine adrenal medulla served as positive control tissue for synaptophysin. The other antibodies had internal controls: for GFAP this were nerves in the larger portal tracts, while for α-SMA, desmin, HHF35 and vimentin arterial smooth muscle cells served as positive control tissue.

For TEM, additional liver samples were taken from two female dogs, three and seven years old. Both were normal control animals euthanized for liver-unrelated research projects. Projects were approved by the responsible ethical committees for the use of experimental animals as required under Dutch legislation. After euthanasia as part of the projects, we were allowed to take liver tissue. Liver samples were taken immediately postmortem, fixed in 4% paraformaldehyde for 2 days, subsequently washed and transferred in methanol to an auto freezing device from Reichert. Freeze substitution was performed 36 h at -85°C in methanol, temperature was gradually raised in 5°C-steps to -45°C, followed by serial substitution from methanol to Lowicryl HM20 from methanol:HM20 = 2:1 (2 × 1 h) to methanol:HM20 = 1:2 (2 × 1 h) and pure HM20 2 h at -45°C. Polymerisation was performed for 36 h at -45°C, then temperature was raised in 5°C-steps for 13 h up to 20°C. Temperature stayed 20°C for 150 h all under UV-light. After ultrathin sectioning grids were labelled according to the procedure for single labelling. Free aldehyde groups were blocked in 50 mM glycine in PBS for 15 min, followed by 30 min aurion blocking solution for goat gold conjugates and washed in BSA-c buffer (PBS + 0.1% BSA-c, pH 7.4), 3 × 5 min. Overnight incubation of the primary antibodies α-SMA and HHF35 diluted in BSA-c buffer (1:1200 and 1:300 respectively) was followed by BSA-c buffer wash (6 × 5 min) and incubation of goat-anti-mouse IgG ultra small gold diluted 1:50 in BSA-c buffer (2 h). BSA-c buffer wash (6 × 5 min) and PBS wash (3 × 5 min) was done previous to postfixation in 2% glutaraldehyde in PBS (5 min), followed by wash in PBS (5 min) and distilled water (5 × 2 min). Signal enhancement was done using Aurion R-Gent SE-EM (30 min), and subsequent washing in distilled water (5 × 2 min). Grids were then stained with uranyl acetate and lead citrate. For sample evaluation a Philips CM 10 TEM was used.